Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels were first discovered in photoreceptors where they shape the light response. They exhibit several properties that make them specialized for retinal signaling: 1) they are weakly K+ selective, 2) they are activated by membrane hyperpolarization, instead of depolarization seen in virtually every other voltage-gated channel, and 3) they are regulated by the direct binding of cyclic nucleotides to an intracellular domain. Our long term goal is to understand the molecular mechanisms for these properties to better understand the physiology and pathophysiology of the channels in the brain and heart. In previous funding periods we have made great progress toward achieving this goal. We have solved the molecular structures of HCN and related channels and invented ground- breaking new fluorescence methods that allow us to record molecular rearrangements in intact channels simultaneous with electrophysiological recording. In this funding period, we propose to apply these methods to determine the molecular mechanisms of hyperpolarization activation and cyclic nucleotide modulation. These experiments will lead to the first dynamic picture for how HCN channels regulate the excitability of neurons and cardiomyocytes.